Cyclodextrin grafted biocompatible amphilphilic polymer and methods of preparation and use thereof

ABSTRACT

Amphiphilic biocompatible cyclodextrin grafted polymers comprising a hydrophobically modified cyclodextrin moiety, a linear linker and a biocompatible hydrophilic polymer backbone, wherein said cyclodextrin moiety is grafted to said biocompatible hydrophilic polymer backbone by said linker are disclosed. The cyclodextrin-grafted biocompatible polymers of this invention may be used as bioactive agent carriers. Methods of making and using such cyclodextrin-grafted biocompatible polymers are disclosed.

BACKGROUND OF THE INVENTION

[0001] This invention relates to novel polymeric bioactive agentcarriers. More particularly, the invention relates tocyclodextrin-grafted biocompatible polymers used as bioactive agentcarriers and methods of making thereof.

[0002] Many biologically active molecules such as anti-viral agents,anti-cancer agents, peptides/proteins and DNA, effective for a varietyof therapeutic applications, have become commercially available throughadvances in recombinant DNA and other technologies. However, an idealcarrier for drugs and active agents is always needed to facilitate theirsolubility, delivery and effectiveness.

[0003] Cyclodextrins (CDs) are cyclic oligosaccharides, usuallyconsisting of six to eight glucose units, which have a truncated coneshape with the wide open side being formed by secondary hydroxyl groups(2-OHs and 3-OHs) and the narrower side by primary hydroxyl groups(6-OHs). Cyclodextrins provide for unique micro-heterogenousenvironments since the exterior of the molecule is hydrophilic while thecavity is hydrophobic due to the relatively high electron density. Theinclusion properties of cyclodextrins, namely, complex-formation betweena guest molecule and a cyclodextrin molecule, have been extensivelyinvestigated. The complexes, which are formed in the solid state and insolution, consist of guest molecules which are held in the cavity of thehost cyclodextrin and are stabilized by Van der Waals forces, and, to alesser extent, by dipole-dipole interactions. Inclusion complexes inaqueous solutions are thought to be further stabilized by hydrophobicinteractions, i.e., by the tendency of solvent water to push hydrophobicsolutes of suitable size and shape into the essentially hydrophobiccavity, in order to attain the “most probable structure” of the solventand obtain minimal energy in the overall system.

[0004] Practical use of natural cyclodextrins (α-, β-, and γ-CDs) asdrug carriers is restricted by their low aqueous solubility. Safety isanother major concern of cyclodextrins being used as drug carriers dueto the toxicity of CD. Modification of the parent cyclodextrin toimprove safety while maintaining the ability to form inclusion complexeswith various substrates has been the goal of numerous research groups.Some groups have also focused on improving interaction between thepharmaceuticals and the cyclodextrins while others have attempted toprepare materials that can be chemically defined more precisely.

[0005] The two most promising cyclodextrin derivatives which aresuitable for parenteral administration are hydoxylpropyl β-cyclodextrin(HPβCD or HPCD) and sulfobutylether-β-cyclodextrin (SBEβCD or SBE-CD).HPβCD has generally been found to be safe when administered parenterallyin animals and humans [Pitha et al, J Pharm Sci, 84 (8), 927-32 (1995)].Minor reversible histological changes have been observed in high doseanimal studies (100-400 mg/kg) and more significant hematologicalchanges were observed in these high dose studies suggesting red bloodcell damage had occurred. No adverse effects were observed in humanstudies. SBEβCD has also been found to be safe when administeredparenterally in mice [Rajewski et al, J Pharm Sci, 84 (8), 927-32(1995)]. However, like most of the modified cyclodextrins, the bindingconstant between drugs and HPβCDs is usually less than those with theparent or unmodified cyclodextrin. Due to steric hindrance of the hostmolecule, the higher the degree of hydroxylpropyl substitution thepoorer the drug binding.

[0006] Hydrophobic modifications of cyclodextrins have also beenprepared in attempts to improve the formulations of some CDinclusionable drugs. It was found that partial methylation of thehydroxyl groups at the 2- and 6-position of β-cyclodextrin (DM-βCD orDMCD) generally leads to stronger drug binding due to increasedhydrophobic interactions. Although the methylated cyclodextrins arehighly water soluble, they also have greater toxicity. The toxicity ofDMβCD was reduced significantly by modifying the free 3-hydroxyl groupswith acetyl groups. This indicates that water-soluble cyclodextrinderivatives with superior bioadaptability and inclusion ability can beprepared by carefully selecting the substitution groups. Controlling thedegree of substitution is also important in balancing water solubilityand complexing capability. When the substitution groups are morehydrophobic than methyl groups, such as an ethyl group, an acetyl group,etc., the whole cyclodextrin derivative becomes practically waterinsoluble. These compounds have been shown to have potential applicationas sustained release carriers for water-soluble drugs. Among thealkylated cyclodextrins, heptakis(2,6-di-O-ethyl)-β-cyclodextrin andheptakis(2,3,6-tri-ethy)-β-cyclodextrin were the first slow-releasecarriers to be used in conjunction with water soluble diltiazem,isosorbide dinitrate, and the peptide buserelin acetate.

[0007] On the other hand, the peracylated cyclodextrins with mediumalkyl chain lengths (C₄-C₅) are particularly useful as novel hydrophobiccarriers due to their multifunctional and bioadaptable properties. Theyhave broad applicability for various routes of administration: forexample, the bioadhesive properties ofheptakis(2,3,6-tri-O-butanoyl-β-cyclodextrin (C₄) can be used in oraland transmucosal formulations, while the film-forming properties ofheptakis (2,3,6-tri-O-valeryl)-b-cyclodextrin (C₅) are useful intransdermal preparations. In oral applications, the release ofmolsidomine, a water-soluble and short-half life drug, was markedlyretarded by complexation with peracylated-β-cyclodextrins in decreasingorder of their solubility, particularly by those having carbon chainslonger than the butylated derivatives. When the complexes wereadministered orally to beagle dogs,heptakis(2,3,6-tri-O-butanoyl)-β-cyclodextrin suppressed the peak plasmalevel of molsidomine and maintained a sufficient drug level for a longperiod, while use of other derivatives having shorter or longer chainsthan heptakis(2,3,6-tri-O-butanoyl)-β-cyclodextrin proved to beinsufficient. This indicates thatheptakis(2,3,6-tri-O-butanoyl)-β-cyclodextrin may be a useful carrierfor orally administered water-soluble drugs, especially for drugs whichare metabolized in the GI tract. The superior and sustained effectexhibited with the heptakis (2,3,6-tri-O-butanoyl)-β-cyclodextrin may bea result of both increased hydrophobicity and mucoadhesive properties.Because of its hydrophobicity,heptakis(2,3,6-tri-O-butanoyl)-β-cyclodextrin, as well as otherhydrophobic cyclodextrin derivatives, can only be used in solid or oilyformulations. On the other hand, like natural β-cyclodextrin, theirmembrane toxicity, which causes tissue irritation and hemolysis in aconcentration-dependent manner is another limitation of theirpharmaceutical application. For example, the concentration of DM-β-CDthat induces 50% hemolysis of human erythrocytes is lower than that ofso called bioadaptable CD derivatives such as 2-hydroxypropyl-β-CD,sulfobutyl ether of β-CD, and maltosyl-β-CD. The hemolytic activity ofcyclodextrins is associated with the extraction of membrane components,mainly through inclusion action with cholesterol. However, this drawbackcan be overcome by further structural modification of alkylated CDs, forexample, heptakis(2,6-di-O-methyl-3-O-acetyl)-β-CD (DMA-β-CD) was foundto have much weaker hemolytic activity while keeping a similar inclusionability to that of DM-β-CD [Hirayama et al, J Pharm Sci, 88 (10), 970-5(1999)]. Since cyclodextrins are poorly adsorbed from the GI tractfollowing oral administration, the oral administration of cyclodextrinsraises minimal safety concerns that may result from the systemicabsorption of the cyclodextrins themselves. However, cyclodextrins maycause secondary systemic effects through increased GI elimination ofcertain nutrients and bile acids. This effect is most notable for {acuteover (υ)}-cyclodextrin assisted fecal elimination of bile acids. Theincreased elimination, however, was only observed at very high oraldoses of cyclodextrin (up to 20% of diet). The secondary effects of theincreased bile acid elimination are increased conversion of serumcholesterol to the bile acids with subsequent lowering of plasmacholesterol levels.

[0008] For years, various kinds of cyclodextrins have been prepared toimprove the physicochemical properties and inclusion capabilities ofparent cyclodextrins, and some of the pharmaceutical products containingcyclodextrins have been approved. Because large amounts of cyclodextrinsare necessary to alter the solubility properties of the drugs beingcarried, the toxicity of the cyclodextrin needs to be very low in orderto safely delivery the necessary dose of a drug. Therefore eitherreducing the total dose or reducing the intrinsic toxicity ofcyclodextrins can widen the pharmaceutical applications ofcyclodextrins.

[0009] In view of the foregoing, it will be appreciated that providingimproved cyclodextrin containing bioactive agent carriers and a methodof using them would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides a new class of amphiphiliccyclodextrin containing polymers wherein multiple hydrophobiccyclodextrin or derivitized cyclodextrin moieties are conjugated with orgrafted to a biocompatible hydrophilic polymeric backbone, throughappropriate biodegradable or non-biodegradable linkers. Optionally, oneor more or a mixture of targeting moieties (TM) may also be covalentlybound to the polymeric backbone. The CD-grafted polymers of the presentinvention can be synthesized by coupling two to thirty CDs orderivatives thereof to a hydrophilic polymer, i.e. a polyethylene glycol(PEG) or poly N-(2-hydroxylpropyl)methacrylamide) (HPMA), via a properlinker. If desired, as described above, one or more targetingmoieties(TM) may optionally be covalently attached to the polymerbackbone. The purpose of using the targeting moiety is to targetparticular cells for drug delivery. The synthesized carrier, namely ahydrophobic CD-grafted hydrophilic polymer, results in better solubilityand reduced cytotoxicity of the drug/carrier complex.

DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0011]FIG. 1 is a graphic illustration showing the stability ofPaclitaxel/CD complexes in 50% serum or 10×PBS dilutions.

[0012]FIG. 2 depicts a reaction scheme for synthesis of PEG-SS-AcCD

[0013]FIG. 3 depicts a reaction scheme for synthesis of PEG-SS-DECD

[0014]FIG. 4 depicts a reaction scheme for synthesis of PEG-GFLG-DECD

[0015]FIG. 5 depicts a reaction scheme for synthesis of PEG-C3-AcCD,PEG-C3-DECD and PEG-C3-BnCD.

[0016]FIG. 6 depicts a reaction scheme for synthesis of PEG-L8-AcCD,PEG-L8-DECD.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Before the present composition and method for drug delivery aredisclosed and described, it is to be understood that this invention isnot limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

[0018] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

[0019] “Active agents” refers to those agents that can function as guestmolecules of the instant invention. Active agents include chemicals andother substances which can form an inclusion complex with a cyclodextrinor derivatized cyclodextrin grafted polymer and are inhibitory,antimetabolic or preventive toward any disease (i.e. cancer, syphilis,gonorrhea, influenza and heart disease) or inhibitory or toxic towardany disease causing agent. Active agents include numerous drugs such asanticancer drugs, antineoplastic drugs, antifungal drugs, antibacterialdrugs, antiviral drugs, cardiac drugs, neurological drugs, and drugs ofabuse; alkaloids (i.e. camptothecins), antibiotics, bioactive peptides,steroids, steroid hormones, polypeptide hormones, interferons,interleukins, narcotics, nucleic acids including antisenseoligonucleotides, pesticides and prostaglandins. Active agents alsoinclude aflatoxins, ricins, bungarotoxins, irinotecan, ganciclovir,furosemide, indomethacin, chlorpromazine, methotrexate, cevinederivatives and analogs including cevadines, desatrines, veratridine. Italso includes various flavone derivatives and analogs includingdihydroxyflavones (chrysins), trihydroxyflavones (apigenins),pentahydroxyflavones (morins), hexahydroxyflavones (myricetins),flavyliums, quercetins, fisetins; various antibiotics includingpenicillin derivatives (i.e. ampicillin), anthracyclines (i.e.doxorubicin, daunorubicin), teramycins, tetracyclines,chlorotetracyclines, clomocyclines, butoconazole, ellipticines,guamecyclines, macrolides (i.e. amphotericins), filipins, fungichromins,nystatins; various purine and pyrimidine derivatives and analogsincluding 5′-fluorouracil, 5′-fluoro-2′-deoxyuridine, and allopurinol;various photosensitizer substances, especially those used for singletand triplet oxygen formation useful for photodynamic, phthalocyanine,porphyrins and their derivatives and analogs; various steroidderivatives and analogs including cholesterols, digoxigenins; variouscoumarin derivatives and analogs including dihydroxycoumarins(esculetins), dicumarols; chrysarobins, chrysophanic acids, emodins,secalonic acids; various dopas, derivatives and analogs including dopas,dopamines, epinephrines, and norepinephrines (arterenols).

[0020] “Parenteral” shall mean intramuscular, intraperitoneal,intra-abdominal, subcutaneous, and, to the extent feasible, intravenousand intraarterial.

[0021] “Biocompatible” means that the substance is nonimmunogenic,nonallergenic and will cause minimum undesired physiological reaction.They may be degraded biologically and they are “biologically neutral” inthat they lack specific binding properties or biorecognition properties.

[0022] “Linkers” or “linkages” are defined as types of specific chemicalmoieties or groups used within the chemical substances that covalentlycouple the cyclodextrin moiety to the polymer backbone and may be eitherbiodegradable or non-biodegradable. Suitable linkers are morespecifically defined below.

[0023] “Drug” shall mean any organic or inorganic compound or substancehaving bioactivity and adapted or used for a therapeutic purpose.Proteins, hormones, anti-cancer agents, oligonucleotides, DNA, RNA andgene therapies are included under the broader definition of drug.

[0024] “Peptide,” “polypeptide,” “oligopeptide” and “protein” shall beused interchangeably when referring to peptide or protein drugs andshall not be limited as to any particular molecular weight, peptidesequence or length, field of bioactivity or therapeutic use unlessspecifically stated.

[0025] “Targeting moiety” refers to those moieties that bind to aspecific biological substance or site. The biological substance or siteis considered the “target” of the targeting moiety that binds to it.Examples of suitable targeting moieties are described below. Examples ofsuitable targeting moieties includes antigens, haptens, biotin, biotinderivatives, lectins, galactosamine and fucosylamine moieties,receptors, substrates, coenzymes, cofactors, proteins, histones,hormones, vitamins, steroids, prostaglandins, synthetic or naturalpolypeptides, carbohydrates, lipids, antibiotics, drugs, digoxins,pesticides, narcotics, neuro-transmitters, and various nucleic acids.

[0026] A “nucleic acid” is defined as any nucleic acid sequence from anysource. The nucleic acid includes all types of RNA, DNA, andoligonucleotides including probes and primers used in polymerase chainreaction (PCR) or DNA sequencing, antisense oligonucleotides andphosphorthioate oligonucleotides. Also included are synthetic nucleicacid polymers, such as methylphosphonate oligonucleotides,phosphotriester oligonucleotides, mopholino-DNA and peptide nucleicacids (PNA) including PNA clamps, DNA and/or RNA fragments, andderivatives from any tissues, cells, nuclei, chromosomes, cytoplasm,mitochondria, ribosomes, and other cellular sources.

[0027] A “cyclodextrin (CD)”, is a cyclic oligosaccharide composed ofglucose monomers coupled together to form a conical, hollow moleculewith a hydrophobic interior or cavity. The cyclodextrins of the instantinvention can be any suitable cyclodextrin, including alpha-, beta-, andgamma-cyclodextrins, and their combinations, analogs, isomers, andderivatives. Cyclodextrins can be either natural or modified withhydrophobic groups as will be described in greater detail below.

[0028] In describing this invention, references to a cyclodextrin“complex”, means a noncovalent inclusion complex. An inclusion complexis defined herein as a cyclodextrin or derivatized cyclodextrinfunctioning as a “host” molecule, combined with one or more “guest”molecules that are contained or bound, wholly or partially, within thehydrophobic cavity of the cyclodextrin or its derivative. Most preferredCDs are derivatives such as carboxymethyl CD, glucosyl CD, maltosyl CD,hydroxypropyl cyclodextrins (HPCD), 2-hydroxypropyl cyclodextrins,2,3-dihydroxypropyl cyclodextrins (DHPCD), sulfobutylether CD, acylated,ethylated and methylated cyclodextrins. Also preferred are oxidizedcyclodextrins that provide aldehydes and any oxidized forms of anyderivatives that provide aldehydes. Also included are altered forms,such as crown ether-like compounds and higher homologues ofcyclodextrins.

[0029] “Controlled release” is defined as the release of a capturedguest molecule/drug from the CD polymer carrier only by cleavage ofcertain linkages that were used to synthesize the carrier.

[0030] This invention relates to novel CD-grafted biocompatibleamphiphilic polymers and the methods of preparation thereof for use asbioactive agent carriers. The invention, in one of its most generaldefinitions, concerns a complex between a bioactive agent and at leastone CD-grafted polymeric conjugate comprising a biocompatiblehydrophilic polymer backbone such as PEG and HPMA, poly-L-Lysine (PLL)and polyethylenimine (PEI) which is grafted with at least one, andpreferably a multiplicity, of hydrophobically modified CDs. Optionally atargeting moiety (TM) may be covalently linked to the polymeric carrier.

[0031] The preferred cyclodextrin containing polymers may be defined bya cyclodextrin containing polymer wherein cyclodextrin or derivatizedcyclodextrin moieties are connected to a biocompatible hydrophilicpolymer backbone by a single spacer arm to the 2, 3, or 6-position ofthe cyclodextrin which can be represented by Formula 1 as follows:

[0032] (1) P is a biocompatible hydrophilic polymer backbone having amolecular weight range from 2,000 to 1,000,000 Daltons, preferably 5,000to 70,000 Daltons, and most preferably 20,000 to 40,000 Daltons.Preferably the biocompatible polymer backbone is a hydrophilic polymerselected from the group consisting of polyethylene glycol (PEG),N-(2-hydroxypropyl)methacrylamide polymer (HPMA), polyethylenimine (PEI)and polylysine (pLL) which are appropriately endcapped as is known inthe prior art and which also may be substituted with substituents thatdo not adversely affect the functionality of the polymer for itsintended purpose. Preferably biocompatible polymer backbone is apolyethylene glycol (PEG) polymer. When the cyclodextrin is attached atthe 2, 3 or 6 position of the cyclodextrin the corresponding R₁O-, R₂O-or R₃O-group will be replaced and the 2-, 3- or 6-carbon of theglucopyranose will be covalently attached to linker X;

[0033] (2) R′ is a member selected from the group consisting ofhydrogen, a tissue targeting moiety (TM) or a cell membrane fusionmoiety (FM) as described herein with the proviso that a mixture ofhydrogen, targeting moieties and cell fusion moieties may be found onthe same polymer backbone and/or within the polymer composition;

[0034] (3) X is a linker having the formula:

-Q-Z-Q′-

[0035] where Q is covalently bonded to the hydrophilic polymer chaineither directly or by means of a pendant alkyl or other functional groupand Q′ is covalently bonded to the cyclodextrin. Q and Q′ areindependently members selected from the group consisting of NR₄, S, O,CO, CONH, and COO. In other words Q and Q′ can comprise amine,alkylamine, acylamine, thio, ether, carbonyl, amide or ester moieties. Zcomprises a member selected from the group consisting of an alkylenedisulfide, [—(CH₂)_(a)S—S(CH₂)_(a)—], alkylene [—(CH₂)_(a)—], alkyleneoxide (—[(CH₂)_(a)O]_(b)(CH₂)_(a)—), or a short chained peptide where ais an integer of 1 to 10 and b is an integer of 1 to 20. Preferably Q isan amide and Q′ is an amine, alkyl amine or acyl amine and the linkerhas the formula: —CONH-Z-NR₄—. Most preferably Q will be attached to aderivatized polymer chain through an alkylene (—CH₂—)_(a) group. When Zis an alkylene disulfide, alkylene oxide or peptide, the linker isbiodegradable. When Z is alkylene, the linker is non-biodegradable;

[0036] (4) R₁, R₂, R₃ and R₄ are independently members selected from thegroup consisting of H, alkyl (C_(n′)H_(2n′+1)), alkenyl(C_(n′+1)H_(2(n′+1)−1)) or acyl (C_(n′)H_(2n′+1)CO) where n′ is aninteger of 1 to 16, preferably 1 to 8, most preferably 1 to 4. When R₁,R₂, R₃ and R₄ are H, the cyclodextrin is more hydrophilic in nature.When one or more of R₁, R₂, R₃ and R₄ are alkyl, alkenyl or acyl groups,the derivatized cyclodextrin becomes more hydrophobic in nature.Therefore, when each of R₁, R₂, R₃ and R₄ is alkyl, alkenyl or acyl, thecyclodextrin is most hydrophobic. The acyl derivatized cyclodextrins aremore biodegradable than the alkyl or alkenyl derivatized cyclodextrins;

[0037] (5) q is an integer of 5, 6 or 7, which makes the pendantcyclodextrin moiety to be α-, β-, or γ-cyclodextrin derivative,respectively. Preferably q is 6 or 7, and most preferably q is 6. Inother words, the preferred cyclodextrin is a β-cyclodextrin;

[0038] (6) w is an integer such that each polymer backbone containsbetween 1.5 and 30 and preferably between 2 and 15 cyclodextrin moietiesper 20 KD of polymer backbone. The integer “w” represents an average ofcyclodextrin moieties in a polymeric composition since a polymericcomposition is a mixture of polymer chains where each polymer in thechain may be variable in length, molecular weight and number ofcyclodextrin moieties. Hence, each polymer has a weight averagemolecular weight and an average number of cyclodextrin moieties per 20KD of polymer backbone within such polymeric composition.

[0039] One embodiment of the present invention is a new class ofCD-grafted-biocompatible polyethylene glycol (PEG) polymer which can berepresented by Formula 2 as follows:

[0040] where q, w, X, R, R₁, R₂, R₃ and R₄ are as described in Formula1, m and n are integers sufficient that when combined with w theyrepresent a polyethylene oxide polymeric chain having the molecularweight as described for the hydrophilic polymer in Formula 1. In otherwords, as noted in Formula 1, the molecular weight of the biocompatiblepolyethylene oxide hydrophilic polymer backbone is preferably within therange of 5,000 to 1,000,000, more preferably within the range of 5,000to 70,000 and most preferably within a range of 20,000 to 40,000. Asnoted in reference to Formula 1, the CDs can be grafted to the polymerby a single arm linker X via the 2, 3 or 6 positions of the CD moleculeand, preferably, are grafted via 6 position of the CD molecule. While whas the same numerical value as in Formula 1 it is to be noted that w isused to denote the number of cyclodextrin units per 20K of polymerbackbone and does not refer to a polymeric unit containing “w”consecutively joined polyethylene glycol (CH₂CHXO) monomers. In otherwords, the polymer backbone contains “w” monomer units containing apendent cyclodextrin which are spaced along the polymer backbone. Thespacing may be random or uniform depending upon the synthesis.

[0041] Most preferably, the cyclodextrin containing polymers, arepolyethylene glycol polymer backbones containing pendant CDs havingfollowing Formula 3 as follows:

[0042] where Q, Q′, Z, R, R₁, R₂, R₃, R₄, a and q are as described inFormula 1, w is an integer such as to provide between 1.5 and 30cyclodextrin units, and preferably between 2 and 15 cyclodextrin unitsper 20 KD polymer chain, as an average, m and n is integers sufficientthat when combined with w they represent a polyethylene oxide polymericchain having the molecular weight as described for the hydrophilicpolymer in Formula 1. As explained for Formula 2, the monomericpolyethylene glycol units containing the pendent cyclodextrin are notconsecutively joined and may be randomly or uniformly spaced along thepolymer backbone.

[0043] Specific β-cyclodextrin co-polymers falling within the scope ofFormula 3 are listed in Table 1. TABLE 1 CD Comp. Polymer No. ID w Q ZQ′ R₁ R₂ R₃ R₄  6 PEG- 5 C(O)NH SS NR₄ H H H H SS-CD 13 PEG- 4.5 C(O)NHC3 NR₄ H H H H C3-CD 18 (a) PEG- 5.5 C(O)NH L8 NR₄ H H H H 18 (b) L8-CD8.5  7 PEG- 1.5 C(O)NH SS NR₄ C₂H₅ H C₂H₅ C₂H₅ SS- DECD 11 PEG- 4.5C(O)NH GFLG NR₄ C₂H₅ H C₂H₅ C₂H₅ GFLG- DECD 14 PEG- 2.6 C(O)NH C3 NR₄C₂H₅ H C₂H₅ C₂H₅ C3- DECD 20 PEG- 3.9 C(O)NH L8 NR₄ C₂H₅ H C₂H₅ C₂H₅ L8-DECD  3 PEG- 5 C(O)NH SS NR₄ CH₃CO CH₃CO CH₃CO CH₃CO SS- AcCD 15 PEG-4.5 C(O)NH C3 NR₄ CH₃CO CH₃CO CH₃CO CH₃CO C3- AcCD 19 (a) PEG- 5.5C(O)NH L8 NR₄ CH₃CO CH₃CO CH₃CO CH₃CO 19 (b) L8- 8.5 AcCD 16 PEG- 4.5C(O)NH C3 NR₄ C₃H₇CO C₃H₇CO C₃H₇CO C₃H₇CO C3- BnCD

[0044] In Table 1 SS is —CH₂CH₂SSCH₂CH₂—, C3 is —CH₂CH₂CH₂—, L8 is—CH₂CH₂OCH₂CH₂OCH₂CH₂— and GFLG is the tetrapeptide Gly-Phe-Glu-Gly.

[0045] These novel CD-grafted polymers of the present invention have thefollowing advantages over their monomer precursors as drug carriers.

[0046] First, they have increased water solubility and reduced toxicity.Polyethylene glycol (PEG) is a linear polyether diol with many usefulproperties, such as good solubility, biocompatibility due to minimaltoxicity, immunogenicity, and antigenicity, and good excretion kinetics.These features have made PEG the most extensively studied drug carrierin pharmaceutical research which eventually lead to its FDA approval forinternal administration. Therefore PEG can change the physical-chemicalproperties and toxicities of conjugated cyclodextrins to make them morebiocompatible.

[0047] In addition, these CD-grafted polymers also provide enhancedguest molecule binding stability. Hydrophobic modification of CDsprovides for a more hydrophobic interior and exterior of thecyclodextrin cavity and so increases the stability of inclusioncomplexes. Moreover multiple CDs in one polymer backbone will increaselocal CD concentration and produce cooperation in drug binding.Therefore, an amphiphilic co-polymer may form a polymeric micelle afterbinding to appropriate guest drugs through extra hydrophobicinteractions or ionic interactions. Furthermore, these drugs containingCD-grafted polymers can be absorbed by cells through pinocytosis ratherthan by passive diffusion.

[0048] Moreover, the CD-grafted polymer can be used for thecontrolled-release and targeted-delivery of a bioactive agent. Thepolymer is likely to form a special type of polymeric micelles withappropriate drugs. Passive drug targeting can increase drug efficiencyby targeting specific cells or organs, therefore reducing accumulationof the drug in healthy tissues and minimizing its toxicity therebyallowing higher doses to be administered, if needed. Followingintravenous administration, polymeric micelles have been found to have aprolonged systemic circulation time due to their small size andhydrophilic shell, which minimizes uptake by the mononuclear phagocytesystem (MPS), and to their high molecular weight which prevents renalexcretion. Polymeric micelle-incorporated drugs may accumulate in tumorsto a greater extent than the free drug and show reduced distributioninto non-targeted areas such as the heart [Kwon et al, J Control Rel,29, 17-23 (1994)]. Accumulation of polymeric micelles in malignant orinflamed tissues may be due to increased vascular permeability andimpaired lymphatic drainage (enhanced permeability and retention (EPR)effect. The EPR effect is considered as a passive targeting method, butdrug targeting could be further increased by binding to targetingmoieties such as antibodies or sugars or by introducing a polymersensitive to variation in temperature or pH. Targeting micelles or pHsensitive micelles can serve for the delivery of drug to tumors,inflamed tissues or endosomal compartments, since they all areassociated with a lower pH than normal tissue [Litzinger et al, BiochimBiophys Acta, 1113 (2), 201-27 (1992); Tannock et al, Cancer Research,49 (16), 4373-84 (1989); Helmlinger et al, Nat Med 3 (2), 177-82(1997)].

[0049] PEG is commercially available in a variety of molecular masses atlow dispersity (Mw/Mn<1.1). Based on their molecular size, they arearbitrarily classified into low molecular weight PEG (Mw<20,000) andhigh molecular weight PEG (Mw>20,000). Most recent applications of PEGare focused on the attachment of cytotoxic anticancer drugs to the PEGor the grafting of PEG to proteins, micelles or liposomes which leads toa reduction in systemic toxicity, longer retention time within the body,alteration in biological distribution, and improvements in therapeuticefficacy [Takakura et al, Crit Rev Oncol, Hematol 18(3), 207-31 (1995);Duncan et al, Anticancer Drugs, 3 (3), 175-210 (1992)]. A recent studyfound that the renal clearance of PEG decreased with an increase inmolecular weight, with the most dramatic change occurring at a MW of30,000 after i.v. administration. The half-time (t1/2) of PEGcirculating in blood also showed a concomitant and dramatic increase.For instance, the t1/2 for PEG went from approximately 18 min to 16.5hour as the molecular weight increased from 6,000 to 50,000.Consequently, conjugation of anticancer drugs with PEG of a molecularweight of 20,000 or greater can prevent rapid elimination of thePEG-conjugated species and allow for passive tumor accumulation[Greenwald et al, Crit Rev Ther Drug Carrier Syst 17 (2), 101-61(2000)].

[0050] In one embodiment of the present invention, a carboxyl groupgrafted PEG (20,000 Daltons or 25,000 Daltons containing 8 to 10carboxyl groups per PEG molecule) is used as the starting material toconjugate with the cyclodextrins. In order to keep the steric hindranceeffect to a minimum, CD moieties were conjugated at the small open end(6-position) of their cavity to the PEG backbone through one of the 7primary hydroxyl groups. In addition, a flexible linear linker was usedto keep the CD moiety away from the polymer backbone and allow it tomove freely. Due to the biocompatibility of the materials and pliabilityof the polymers of the present invention, they will cause minimaltoxicity and minimal mechanical irritation to the surrounding tissue.

[0051] A dosage form comprised of a solution of the grafted polymer thatcontains either dissolved drug or drug as a suspension or emulsion isadministered to the body. The only limitation as to how much drug can beloaded into the formulation is one of functionality, namely, the drugload may be increased until the desired properties of the polymer areadversely affected to an unacceptable degree, or until the properties ofthe formulation are adversely affected to such a degree as to makeadministration of the formulation unacceptably difficult. Generallyspeaking, it is anticipated that in most instances the drug will make upbetween about 0.01% to 50% by weight of the formulation with ranges ofbetween about 0.1% to 25% being most common. These ranges of drugloading are not limiting to the invention. Provided functionality ismaintained, drug loadings outside of these ranges falls within the scopeof the invention.

[0052] A distinct advantage to the compositions of the subject of thisinvention lies in the ability of the grafted polymer to increase thesolubility and stability of many drug substances. The combination ofhydrophobic CDs and hydrophilic polymers renders the polymer amphiphilicin nature. In that regard it functions much as a combination ofcyclodextrin inclusion and polymeric micelle system. This isparticularly advantageous in the solubilization of hydrophobic or poorlywater soluble drugs such as cyclosporin A, tacrolimus, saquinavir andpaclitaxel.

[0053] Another advantage to the composition of the invention lies in theability of the polymer to increase the chemical stability of many drugsubstances. Various mechanisms for the degradation of drugs have beenobserved to be inhibited when the drug is in the presence of thepolymer. For example, paclitaxel and cyclosporin A are substantiallystabilized in the aqueous polymer composition of the present inventionrelative to certain aqueous solutions of these same drugs in thepresence of organic co-solvents. This stabilization effect on paclitaxeland cyclosporin A is but illustrative of the effect that can be achievedwith many other drug substances.

[0054] The drug loaded CD-grafted polymers of the present invention maybe administered via various routes including parenteral, topical,transdermal, transmucosal, inhaled or inserted into a body cavity suchas by ocular, vaginal, buccal, transurethral, rectal, nasal, oral,pulmonary and aural administration.

[0055] This invention is applicable to bioactive agents and drugs of alltypes including nucleic acids, hormones, anticancer-agents, and itoffers an unusually effective way to deliver polypeptides and proteins.The only limitation to the polypeptide or protein drug which may beutilized is one of functionality. In some instances, the functionalityor physical stability of polypeptides and proteins can also be increasedby addition of various additives to aqueous solutions or suspensions ofthe polypeptide or protein drug. Additives, such as polyols (includingsugars), amino acids, surfactants, polymers, other proteins and certainsalts may be used. Developments in protein engineering may provide thepossibility of increasing the inherent stability of peptides orproteins. While such resultant engineered or modified proteins may beregarded as new entities in regards to regulatory implications, thatdoes not alter their suitability for use in the present invention.

[0056] In addition to peptide or protein based drugs, other drugs fromall therapeutic and medically useful categories may be utilized. Thesedrugs are described in such well-known literature references as theMerck Index, the Physicians Desk Reference, and The PharmacologicalBasis of Therapeutics.

[0057] Paclitaxel is a diterpeniod natural product showing encouragingactivity against ovarian, breast, head, and non-small-cell lung cancers.Recently it has been approved in the form of paclitaxel for treatment ofbreast and refractory human cancers. One of the major problems withpaclitaxel is its extremely low aqueous solubility. The presentformulation of this drug contains 30 mg of paclitaxel in 5 ml of a 50/50mixture of Cremophore EL (polyethoxylated casteror oil, a solubilizingsurfactant) and ethanol. When diluted in saline, as recommended foradministration, the concentration of paclitaxel is 0.6-1.2 mg/ml(0.7-1.4 ml). The diluted solution is expected to contain mixed“micelle” particles of Paclitaxel/Cremophor and has been reported to bephysically unstable over time, because dilution to some concentrationsapparently yields supersaturated solutions. In addition, Cremophor, anon-charged surfactant, has been reported to cause histamine release andto be associated with adverse effects such as severe allergic reactions[Sharma et al, Int J Cancer, 71 (1), 103-7 (1997)]. Cyclodextrinderivatives have been examined to see if they can solubilize paclitaxel.It was found that methylated cyclodextrins worked much better than otherhydrophilic cyclodextrin derivatives in improving the water solubilityof paclitaxel (at 50% CD concentrations, HPCD and DMCD could dissolveabout 0.7 and 33 mg/ml paclitaxel respectively) [Sharma et al. J PharmSci, 84 (10), 1223-30 (1995)]. However, the toxicity of DMCD and thehigh concentration needed to complex therapeutic levels of paclitaxellimit its clinical application. The CD-grafted amphiphilic polymers ofthe present invention provide significant advantages over prior artformulations facilitated by ease of preparation and administration,lowered toxicity, rapid and controlled release of active agents andtargetable delivery.

[0058] Antisense oligonucleotides and their analogs, such as peptide DNA(PNA), morpholino-DNA, P-ethoxy DNA, methylphosphonate-DNA, etc., havebeen shown to have great applications in biomedical research, but theirpharmaceutical applications have been largely limited by their stabilityand/or solubility, and cell uptake behavior. Currently there is noeffective means to safely deliver intact antisense oligonucleotides totheir target sites in vivo. And this is particularly true for theirneutral analogs, such as PNA, morpholino DNA, P-ethoxy DNA andmethylphosphonate-DNA, because they cannot efficiently bind to any ofthe current antisense oligonucleotide carriers which are mostlypoly-cationic polymers. However, the CD-grafted amphiphilic polymers ofthe present invention can be effective carriers of neutral anologsbecause every nucleoside unit has an aromatic base residue which is apotential target to be included by the cyclodextrin, thus the CD-graftedpolymers can bind oligonucleotides and their analogs through enhanced CDinclusion mechanisms. This binding can be very strong due to cooperationbetween the multiple CD moieties on the polymer and the multiplearomatic base rings on antisense oligonucelotides. In addition, extraionic interactions (for charged oligonucleotide) or hydrophobicinteractions (for non-charged oligonucleotide analogs) can alsostrengthen the binding between antisense oligonucelotides and CD-polymercarriers. Eventually the final binding complex may form a loose or tightpolymeric micelle depending on their content, and therefore can safelydeliver antisense oligonucleotides and their neutral analogs to cells.

[0059] In summary, the CD-grafted polymers of the present inventionimprove the drug/binding complex stability via multiple CD moietyco-operations and external hydrophobic or ionic interactions. It islikely that inclusion is an essential mechanism for the drug bindingcapability of the polymers of the present invention. However, ionicinteractions and external hydrophobic interactions (outside the CDcavity) may also make significant contributions depending on themolecular structures of the specific co-polymers and guests.Furthermore, appropriately constructed PEG-CD co-polymers of the presentinvention are excellent paclitaxel solubilizers and carriers for safetherapeutic application. They can also be used as solubilizers andcarriers for other hydrophobic drugs. The CD-grafted amphiphilicpolymers of the present invention are water soluble and biocompatible,and have very slow release kinetics, especially when they contain highweight ratios of hydrophobic moieties. In addition, the strong bindingconstant of the drug/polymer complexes makes for slow release of thebound drug upon dilution, and it sometimes even needs replacement byother molecules. Therefore they may be used as ingredients in oralformulations for delivery of certain water soluble drugs.

[0060] Furthermore, properly constructed CD-grafted polymers of thepresent invention can be used to deliver antisense oligonucleotides andtheir non-charged analogs, as well as hydrophobic peptides and proteins,since external hydrophobic interactions may produce enough stability forhydrophobic antisense oligonucleotides or hydrophobic peptides. Thenegatively charged oligonucleotides are also expected to be good guestmolecules for some specially constructed polymers, because a basicnitrogen in the linker of the polymer could neutralize negative chargeunder appropriate conditions.

[0061] The following Examples are presented to illustrate the process ofpreparing the composition and method of using the composition of thepresent invention.

EXAMPLE 1

[0062] Materials and methods: PEG with pendant propionic acid groups(PEG-10PA and PEG-8PA, Mw=˜20 KD, SunBio, Inc., Anyang City, SouthKorea) was dried overnight in vacuo at room temperature. β-Cyclodextrin(TCI America, Portland, Oreg.) was dried in vacuo at 130° C. overnightbefore use. Other chemicals were from Aldrich Chemical Company, Inc. ofMilwaukee, Wis.) and used as received without further purification. HPLCanalysis was performed on a Waters system equipped with RI detector andUltrahydrogel 120 and Ultrahydrogel 500 SEC columns. ¹H-NMR was recordedon a Varian 400 MHz machine.

Synthesis of PEG-SS-CD (Compound 2)

[0063] Mono-6-(6-amino-3,4-dithio-hexylamino)-6-deoxy-β-cyclodextrin(Compound 1):

[0064] Cystamine dihydrochloride (1.0 g, 4.44 moles, Fw=225.2) wasdissolved in 30 ml distilled water, followed by addition of 1.0 M KOH(8.88 moles) and mono-6-tosyl-β-cyclodextrin (0.5 g, Fw=1289) powder.The resulting suspension was stirred in a 70° C. oil bath overnight,then concentrated to about 4 ml. The mixture was applied on a SephadexG-25 column (2.5×80 cm), eluted with 0.1 M TEA. About 0.38 g compound 1was obtained.

[0065] PEG-SS-CD (Compound 2):

[0066] Carboxyl group grafted PEG (2.24 g, PEG-8PA, 20 kDa, polyethyleneglycol containing 8 pendant propionic acid groups with average molecularweight of ˜20,000) was dissolved in 25 ml anhydrous DMF, the mixture wascooled to 0° C. on ice under protection of argon. To this was added 280ul of tributylamine (1.18 mmoles, Fw=185.36, d=0.778), followed by 175ul of isobutylchloroformate (IBCF, Fw=136.6, d=1.053) in 1 ml DMF. Themixture was stirred at 0° C. for 1 hour. The reaction mixture was thenslowly added to a solution of 1.75 g compound 2 in 100 ml DMF at roomtemperature. After being stirred at room temperature overnight, thereaction was stopped by addition of 1 ml water. The mixture wasconcentrated and then diluted with 60 ml water. The product solution waspurified on a Sephadex G-50 column, eluted with 0.1 M TEA followed byether precipitation. ¹H-NMR analysis indicated that about 5 CD moietieswere conjugated to a PEG backbone having a molecular weight of about20,000 Daltons. The retention time of the product is about 0.45 minutelater than that of the starting PEG as determined by HPLC chromatography[GPC column, Rt (product)=17.33′, vs. Rt (PEG-8A)=16.87′]. ¹H-NMR (400MHz, D2O): δ, 5.0 (s, 7H, H1′), 3.3-3.9 (m, 370H, 41H-CD, 329H-PEG).

EXAMPLE 2 Synthesis of PEG-SS-AcCD (Compound 3)

[0067] PEG-SS-CD (compound 2, 1.0 g, ˜5 CDs/20 kD-PEG) was dried in aP₂O₅ desiccator, followed by co-evaporation with 50 ml anhydrouspyridine. The residue was dissolved in 30 ml pyridine under protectionof argon, followed by addition of 2.0 ml acetic anhydride (Fw=102.1,d=1.08). The mixture was dried on a rotary-evaporator after beingstirred at room temperature for 2 days. The crude product was purifiedby repeated ether precipitation from methanol. HPLC (GPC) analysisshowed a 0.46 minute time delay of the product compared to the startingpolymer (Rt=19.70′ of the product vs. Rt=19.24′ of the reactantpolymer). ¹H-NMR analysis indicates that each 20 kD PEG contains about 5CD moieties and all hydroxyl groups are acetylated. ¹H-NMR (400 MHz,D₂O): δ, 4.7-5.5(s, 14H, H1′, H3′), 3.4-5.5 (m, 382H, 35H-CD, 347H-PEG),2.05 (m, 20H, H-Ac).

EXAMPLE 3 Synthesis of PEG-SS-DECD (Compound 7)

[0068] PEG-SS-NH2 (Compound 4):

[0069] Carboxyl group grafted PEG (PEG-8PA, 2.6 g, 2.0 mmole COOH group)was dissolved in 30 ml anhydrous DMF and cooled to 0° C. on ice. To thiswas added tributylamine (0.35 ml, 1.5 mmoles, Fw=185.36, d=0.778),followed by the addition of isobutyl chloroformate (0.20 ml, 1.5 mmoles,Fw=136.6, d=1.053). The mixture was stirred at 0° C. for 80 minutes andwas carefully added to a solution cystamine (3.5 g, Fw=152.2, 23 mmoles)in 50 ml anhydrous DMF. The mixture was stirred at room temperature for20 hours, concentrated to about 20 ml on rotary evaporator at 40° C.,then dialysed against distilled water (4×5 L over 26 hours, SigmaD-0655, MWCO=12,000) after being diluted with 50 ml water. The dialysissolution was concentrated by rotary evaporation at 40° C., obtaining 4.1g of syrup. The syrup was dissolved in 10 ml methanol, then precipitatedby addition of 80 ml ethyl ether. The precipitate was collected bycentrifugation and this precipitation process was repeated twice. Thefinal product was a white powder, weighing about 2.2 g. The productshowed only one nice peak in its HPLC (GPC) chromatogram, and theretention time (18.66′) was about 1.5 minutes longer than that of thestarting PEG-8PA (17.11′).

[0070] N-(β-Cyclodextrin-6-yl) Glycine Methyl Ester (Compound 5):

[0071] Glycine methyl ester hydrochloride (1.5 g, Fw=125.56, 12 mmoles,from Aldrich) was dissolved in 100 ml anhydrous DMF with protection ofargon. To this was added DIPEA (2.1 ml, 12 mmoles, Fw=129.25, d=0.724),followed by the addition 6-mono-tosyl cyclodextrin powder (3.0 g,Fw=1289, ˜80% pure, ˜1.8 mmoles). The mixture was stirred at roomtemperature to a clear solution. The temperature was slowly raised toabout 70° C. followed by another 4 hour stirring. The mixture was thenconcentrated to a syrup on a rotary evaporator at 55° C. The crudeproduct was dissolved in 40 ml hot water, precipitated by adding ˜80 mlacetone after cooled to room temperature. The white precipitate wascollected by filtration and dried in a vacuum overnight. About 2.3 g ofthe desired compound 5 was obtained. This product was used in next stepwithout further purification.

[0072]N-(Heptakis-2-O-ethyl-6^(B),6^(C),6^(D),6^(E),6^(F),6^(G)-hexa-O-ethyl-β-Cyclodextrin-6^(A)-yl)-glycine(Compound 6):

[0073] N-(β-Cyclodextrin-6-yl) glycine methyl ester (compound 5 about2.0 g, Fw=1206, ˜1.6 mmoles) was dissolved in 15 ml DMSO and 15 ml DMF.The solution was cooled to 0° C. in an ice bath, followed by addition of10 g BaO and 10 g Ba(OH)₂.H₂O with the protection of argon. To thiswhite suspension was slowly added 20 ml diethyl sulfate. The mixture wasstirred at 0° C. for 1 hour, followed by another 24 hour stirring atroom temperature. Another 20 ml of diethyl sulfate was slowly addedwithin an hour, followed by another 24 hour stirring at roomtemperature. To the viscous reaction mixture was slowly added 60 ml 5 NNaOH at 0° C., then the mixture was stirred at room temperature for onehour. It was extracted with 2×200 ml of chloroform. The combined organicphase was concentrated to a wax product after drying with Na₂SO₄. Thecrude product was dissolved in 20 ml methanol, followed by addition of20 ml of distilled water. The mixture was filtered in vacuum to removethe trace amount of precipitate. The clear filtrate was concentrated toget an orange foam solid (about 1.8 g), which contained about 50% of thedesired compound 6. This crude product was directly used in the nextreaction after being dried overnight in vacuum P₂O₅ desiccator.

[0074] PEG-SS-DECD (Compound 7):

[0075] The crude compound 6 (1.4 g, ˜0.46 mmole) was dried byco-evaporation with 2×20 ml anhydrous DMF, then re-dissolved in 20 mlDMF followed by addition of 0.19 ml tributylamine (0.8 mmole, Fw=185.36,d=0.778). The mixture was cooled to 0° C. on ice. To this cold solutionwas slowly added isobutyl chloroformate (60 ul, 0.46 mmole, Fw=136.6,d=1.053) in 2 ml DMF. The mixture was stirred at 0° C. for 1.5 hours,then transferred to a solution of PEG-SS-NH2 (compound 4, 4 g) in 10 mlanhydrous DMF at room temperature, followed by addition of DIPEA (28 ul,0.16 mmole, Fw=129, d=0.724). The mixture was concentrated to a syrupafter overnight stirring at room temperature. The syrup was trituratedwith 30 ml ethyl ether to produce an orange precipitate. The precipitatewas collected by filtration and washed with ethyl ether. The solid wasfurther purified twice by ether precipitation from methanol. About 0.55g light orange solid was obtained. ¹H-NMR indicated the product was thedesired PEG-SS-DECD product, but only about 1.5 CD moieties wereconjugated onto a 20-KD PEG molecule and about 13 ethyl groups percyclodextrin. ¹H-NMR (400 MHz, D2O): δ, 5.1 (7H, m, H1′ and H3′),3.2-3.9 (m, 1041H, 41H-CD, 1000H-PEG), 2.78 (m, 30H, CH2-Et), 1.15 (b,45, CH3-Et).

EXAMPLE 4 Synthesis of PEG-GFLG-DECD (Compound 11)

[0076] Mono-6-(N³-Boc-3-amino-propylamino)-6-deoxy-Cyclodextrin(Compound 8):

[0077] Mono-Boc-1,3-diamino-propane (3.5 g, ˜3.0 moles, Preparedaccording to the methods described by Jean Francois Pons et. al., Eur.J. Org Chem, 1998, 853-859) was dried by co-evaporation with 2.8 ml (12mmoles, Fw=185.36, d=0.778) tributylamine and 30 ml anhydrous DMF twice.The final dried oil was mixed with 100 ml anhydrous DMF, followed byaddition of DIPEA (2.1 ml, 12 mmole, Fw=129, d=0.742). To this solutionwas added 3.5 g of 6-mono-tosyl-6-O-β-cyclodextrin. The mixture wasstirred at room temperature to the complete dissolution of the solid.Then the mixture was stirred overnight at 70° C. in an oil bath. Themixture was concentrated to about 10 ml on a rotary evaporator at 45°C., then precipitated with 100 ml of acetone. The white precipitate wascollected by filtration, washed with acetone. About 3.2 g of product wasobtained. It contained about 60% of the desired compound 8 as estimatedon a TLC (Rf=0.12, Silica gel, developed in 80:10:10/AcOH: CHCl₃: H₂O,stained with 5% phosphomolybdic acid in 95% ethanol). This product wasdirectly ethylated in the next step.

[0078]Mono-(Heptakis-2-O-ethyl-6^(B),6^(C),6^(D),6^(E),6^(F),6^(G)-hexa-O-ethyl-Cyclodextrin-6-yl)-1,3-diamino-propane(Compound 9):

[0079] Mono-6-(N³-Boc-3-amino-propylamino)-6-deoxy-β-Cyclodextrin(Compound 8, 3.0 g) was dissolved in 40 ml anhydrous DMF and 40 ml DMSOat 0° C., then mixed with 10 g of BaO and 10 ml of Ba(OH)₂.H₂O underprotection of argon. The mixture was cooled to 0° C., then 20 ml ofdiethyl sulfate was slowly added. The mixture was stirred at 0° C. for 6hours followed by another 2 days at room temperature. To the reactionmixture was added 25 ml of cold ammonia followed by another 3 hourstirring at room temperature. The final reaction mixture was dilutedwith 50 ml H₂O, extracted with 3×100 ml ethyl acetate. The organic phasewas thoroughly washed with 2×200 ml saturated NaHCO₃ and 3×200 water,then concentrated after drying with sodium sulfate. About 2.8 g oforange solid was obtained after being dried in vacuum overnight. Theproduct was dissolved in 10 ml of trifluoroacetic acid. The clearsolution was stirred at room temperature for 3 hours, then 15 ml ofwater was added. The mixture was stirred at room temperature for another20 minutes, then dried on a rotary evaporator at 45° C. The residue wasdissolved in 150 ml ethyl acetate, washed with 3×100 ml saturated NaHCO₃and 100 ml of saline. The organic phase was concentrated after beingdried with Na₂SO₄. About 2.0 grams of crude compound 9 was obtained.This product was directly used in the next conjugation reaction.

[0080] PEG-GFLF-DECD (Compound 11):

[0081] PEG-GFLG (tetrapeptide Gly-Phe-Leu-Gly grafted PEG polymer,compound 10, ˜4.5 GFLG peptide in a PEG of 20,000 prepared from PEG-8PAand GFLG peptide) (2.0 g, ˜0.4 mmole —COOH, dried by co-evaporation with30 ml DMF) was dissolved in 30 ml anhydrous DMF and 0.17 ml oftributylamine (0.7 mmole, Fw=185.36, d=1.053) with protection of argon.To this was added 0.078 ml (0.6 mmole) isobutylchloroformate in 2 ml DMFafter cooling to 0° C. The mixture was stirred at 0° C. for 1.5 hours,then slowly added to the solution of 2.0 g compound 9 in 20 ml DMF atroom temperature, followed by addition of 0.087 ml of DIPEA (0.5 mmole).The mixture was stirred at room temperature overnight, concentrated toabout 10 ml, precipitated with 90 ml of cold ethyl ether. The orangeprecipitate was collected by filtration and was further precipitated 3times using ether from methanol. The final product was about 2.2 grams.The retention time of the product (Rt=18.42′) was 0.67 minutes longerthan that of the starting PEG-GFLG polymer (Rt=17.76′) on HPLC (GPC)chromatography. ¹H-NMR indicates the product is the desired compound 11:every 20 kD polymer contains about 4.5 tetrapeptide GFLG and 1.8 CDmoieties and every CD moiety has about 13 ethyl groups. ¹H-NMR (400 HMz,D₂O): δ, 7.20 (5H, m, ArH-Phe), 5.1 (2.8H, m, H1′-CD), 3.0-4.0 (645H, m,41H-CD, 574H-PEG, 30H-Et), 1.1 (15.6H,m, 30H, CH3-Et), 0.9 (6H, d,CH3-Leu).

EXAMPLE 5 Synthesis of PEG-C3-AcCD, PEG-C3-DECD and PEG-C3-BnCD

[0082] Mono-6-(γ-amino-propanyl-amino)-6-deoxy-β-cyclodextrin (Compound12):

[0083] Mono-6-tosyl-6-deoxy-cyclodextrin (6.5 g, Fw=1269) was dissolvedin 200 ml of anhydrous DMF and 60 ml of diaminopropane under vigorousstirring at room temperature. The clear mixture was stirred at roomtemperature for 2 hours followed by another 20 hours at 65° C. C. Themixture was concentrated to about 20 ml at 45° C. C. To this was added200 ml of cold isopropanol at room temperature. The white precipitatewas collected by filtration. The solid was re-dissolved in 25 ml waterand 25 ml TEA. To this was slowly added 300 ml of acetone at 0° C. C.The precipitate was collected by filtration, and re-precipitation wasrepeated two-more times. The final product was about 5.5 grams. Itcontains about 80% of the desired compound 12 and about 20% freecyclodextrin. The product was used for the next reaction without furtherpurification.

[0084] PEG-C3-CD (compound 13):Mono-6-(γ-amino-propanyl-amino)-6-deoxy-β-cyclodextrin (compound 12, 6.2g) was conjugated to PEG-8PA (4.1 g) using the same method as describedin the synthesis of PEG-SS-CD. About 4.3 g of pure product was obtainedafter GPC purification. The retention time of the product (17.87′) is0.76 minute longer than that of starting PEG-8PA (17.11′). ¹H-NMRindicates the product is the desired compound 13, which contains about4.5 CD moieties in every 20 KD PEG molecule. ¹H-NMR (400 HMz, D2O): δ,5.0 (7H, s, H1′-CD), 3.4-3.9 (412H, m, 41H-CD, 371H-PEG).

[0085] PEG-C3-AcCD (compound 15): PEG-C3-CD (1.0 gram, ˜4.5 CDs/20 KDPEG) was acetylated using the same method as described in thepreparation of PEG-SS-AcCD. About 1.0 gram of product was obtained andits retention time (17.99′) was only about 7.2 seconds longer than thatof the starting polymer (PEG-C3-CD, 17.87′). However ¹H-NMR indicatedthat the product is the desired compound 15: the polymer contains 4.5 ofCD moieties in every 20 kD PEG and bout 90% of the hydroxyl groups onthe pendent CDs were acetylated. ¹H-NMR (D2O): δ, 4.9-5.4 (14H, m,H1′-CD and H3′-CD), 3.2-4.5 (m, 490H, 35H-CD, 455H-PEG), 2.03 (d, 64H,CH₃CO—).

[0086] PEG-C3-BnCD (compound 16): PEG-C3-CD (Compound 13, 0.9 g, ˜4.5CDs/20 KD PEG) was dried by co-evaporation with 20 ml anhydrous pyridineand then re-dissolved in 30 ml pyridine with protection of argon. Tothis was slowly added 3 ml of butyryl chloride (Fw=106.55, d=1.026) atroom temperature (cooled with ice as the reaction temperature went up).Methanol (5.0 ml) was added after the mixture was stirred at roomtemperature for 4 hours, followed by another 30 minutes of stirring atroom temperature. The mixture was concentrated on a rotary evaporator toa wax solid. The solid was dissolved in 20 ml of methanol, and dilutedwith 20 ml water. The clear solution was dialyzed against 2×5 L 20%isopropanol/water. The opaque dialysis solution was concentrated in aSpeed-Vac at room temperature. The pellet was further precipitated threetimes from methanol using ether. The product is practically insoluble inwater, but very soluble in methanol or chloroform. Yield=90%. ¹H-NMRindicates that the product is the desired compound 16: about 80% of thehydroxyl groups on the pendant cyclodextrins were butyrylated. ¹H-NMR(CDCl₃): δ, 4.6-5.3 (14H, m, H1′ and H3′), 3.2-4.5 (m, 541, 35H-CD,486H-PEG), 2.30 (m, 36H, CH₃CH₂CH₂CO—, 1.65 (m, 36H, CH₃CH₂CH₂CO—), 0.95(m, 54H, CH₃CH₂CH₂CO—).

EXAMPLE 6 Synthesis of PEG-L8-AcCD and PEG-L8-DECD

[0087] Mono-6-(8-amino-3,6-dioxy-octylamino)-6-deoxy-β-cyclodextrin(Compound 17):

[0088] In a 500 ml round bottom flask was charged with2,2′-(ethylenedioxy)bis(ethylamine) (300 ml, Fw=148) andmono-6-tosyl-p-cyclodextrin (24.4 g, Fw=1269, dried in a P₂O₅ desiccatorovernight) under the protection of argon. The suspension was stirred atroom temperature to the complete dissolution of all of the solid (˜1.0hour). The mixture was stirred for another 4 hours at 75° C. Thereaction mixture was slowly poured into 1.8 L of cold isopropanol. Theprecipitate was collected by filtration and washed with isopropanol. Theprecipitate was dissolved in 200 ml warm water (50° C.), then slowlypoured into 1.8 L of ice cold isopropanol with stirring. The precipitatewas collected by filtration after being cooled to −20° C. Thisisopropanol precipitation process was repeated two more times. About 24grams of white powder was obtained. HPLC analysis (GPC, eluted with 0.1M NaNO₃) showed that the product contains about 85% desired compound(Rt=39.21′) and ˜15% non-modified 1-CD (Rt=32.25′), no free diaminereactant was detected. So this product was directly used for nextconjugation. ¹H-NMR (400 HMz, D2O): δ, 4.97 (7H, m, H1′), 3.7-3.9 (26H,m, 7H3′, 7H5′, 6H6′, 6H6″), 3.3-3.6 (24H, m, 7H2/, 7H4′, 1H6′, 1H6″,8H-linker), 2.71 (4H, m, CH₂N-linker).

[0089] PEG-L8-CD (Compound 18):

[0090] PEG-8PA (4.0 g, ˜8-COOH/PEG-20K, ˜1.7 mmoles COOH, dried in aP₂O₅ desiccator overnight and co-evaporated with 50 ml anhydrous DMF)was dissolved in 50 ml anhydrous DMF and 0.54 ml tributyl amine (TBA,Fw=185.36, d=0.778, 2.27 mmoles). The clear mixture was cooled on ice,then 0.29 ml isobutyl chloroformate (IBCF, Fw=136.6, d=1.053, 2.2mmoles) was added at 0° C. The mixture was stirred at 0° C. for 1 hour,and was then slowly added to a solution ofmono-6-(8-amino-3,6-dioxy-octylamino)-6-deoxy-β-cyclodextrin (compound17, 5.0 g , Fw=1336, ˜80% pure, ˜2.6 mmoles, dried in a P₂O₅ desiccatorovernight) in 50 ml anhydrous DMF at room temperature. After overnightstirring, the mixture was concentrated to about 20 ml on a rota-vap at50° C. The mixture was diluted with 60 ml of water and purified on aSephadex-G-50 column (2.5×80 cm, eluted with 0.1 M TEAA, pH=10.0,collected 8 ml/ml). The fractions were analyzed by GPC-HPLC and thepolymer fraction was pooled into two parts: Part A: fraction 9 through30; Part B: fraction 31 through 35.

[0091] Both parts were concentrated to wax solids on rotary evaporatorand then re-dissolved in 15 ml methanol. The products were precipitatedby 5 ml TEA and 120 ml of ethyl ether. The white precipitates werecollected by filtration. Part A and Part B weighed 4.7 gram and 0.55gram, respectively. ¹H-NMR analysis confirmed both parts were thedesired PEG-L8-CD product, but with different cyclodextrin loading: onaverage a 20 KD-PEG polymer contains about 5.5 and 8.5 cyclodextrinmoieties in part A and part B, respectively. ¹H-NMR (400 MHz, D2O): 8,Part A: 5.0 (s, 7H, H1′), 3.3-3.9 (382H, m, 41H-CD, 12H-linker,329H-PEG; Part B: 5.0 (s, 7H, H1′), 3.3-3.9 (256H, m, 41H-CD,12H-linker, 203H-PEG).

[0092] PEG-L8-AcCD (Compound 19):

[0093] PEG-L8-CD (Compound 18, 1.0 g, 5.5 CDs/20 KD PEG, dried in P2O5desiccator overnight) was dried by co-evaporation with 40 ml anhydrouspyridine, then re-dissolved in 40 ml anhydrous pyridine under protectionof argon. To this was added 3.0 ml acetic anhydride. The mixture wasstirred at room temperature for 2 days, concentrated to about 10 ml on arotary evaporator at 45° C. To this was slowly added 90 ml of ethylether. The precipitate was collected by filtration. The product wasfurther purified by ether precipitation three more times from methanol.The final white powder was dried in a vacuum, and it weighed 1.07 g.¹H-NMR confirmed the product is the desired product 19: Every 20 kD PEGcontains about 5.5 CD moieties and about 90% of the hydroxyl groups onthe pendent CD moieties of the polymer were acetylated. ¹H-NMR (D2O): δ,4.9-5.4 (14H, m, H1′-CD and H3′-CD), 3.2-4.5 (m, 422H, 34H-CD,12H-linker, 376H-PEG), 2.03 (d, 64H, CH3CO—).

[0094] PEG-L8-DECD (Compound 20):

[0095] PEG-L8-CD (compound 18, 1.0 g, 5.5 CDs/20 KD PEG, dried in P₂O₅desiccator overnight) was dissolved in 5 ml anhydrous DMSO and 5 mlanhydrous DMF, the solution was cooled to 0° C. on ice under protectionof argon. To this was added 0.75 g BaO and 0.75 g Ba(OH)₂.H₂O powder,immediately followed by addition of 3 ml of diethyl sulfate in threeportions over a one hour period. The suspension was stirred at 0° C. for2 hours, followed by stirring for another 2 days at 4° C. Then 80 ml ofcold ethyl ether was added at 0° C., followed by another 30 minutes ofstirring at 0° C. The orange precipitate was collected by filtration anddissolved in 50 ml 50% methanol/water. The mixture was dialyzed(MWCO=12,000) against 5 L of 0.01 N HCl, then 2×5L water. The finaldialysis solution was concentrated, obtaining about 1 g of wax product.It was further purified by ether precipitation from methanol twice.¹HNMR analysis indicated that about 4 CDs are present in every 20KD-PEG, and each CD moiety carries about 11 ethyl groups. This meansabout 30% of the CD moieties came off the PEG backbone during thealkylation process. ¹H-NMR (400 HMz, D20): δ, 4.9-5.3 (7H, m, H1′-CD),3.1-4.0 (540H, m, 41H-CD, 469H-PEG, 8H-linker, 22H-CH2-ehthyl), 1.2(33H, m, CH3-ethyl)

[0096] Thirteen representative cyclodextrin-grafted-PEG polymers(Table 1) have been prepared according to Examples 1-6 and FIGS. 4-8,wherein the linkers are either biodegradable (X=-SS- or GFLG-) ornon-biodegradable (-C3- or -L8-). The pendent cyclodextrin moieties areeither natural β-CD (PEG-X-CD) or modified with hydrophobic groupsincluding ethyl (PEG-X-DECD), acetyl (PEG-X-AcCD) or butyryl(PEG-C3-BnCD). GPC-HPLC was used to monitor each step of the preparationprocess, and it was found that all final polymer products had longerretention times than the corresponding PEG precursors. The structure ofall of the product polymers were confirmed by ¹H-NMR analysis, it wasfound that their CD contents varied from an average of 1.5 CDs to 8.5CDs on every 20 KD PEG backbone (Table 2). They are all highly solublein most organic solvents (chloroform, methanol, ethanol, etc.). They arealso highly soluble in water, except PEG-C3-BnCD. TABLE 2 Structurecharacteristics of some cyclodextrin grafted PEG co- polymers t_(R)Number of CDs/ Polymer name (min*) 20 Kd Polymer** CD modification**PEG-ss-CD 19.34 3.9 None PEG-ss-AcCD 19.24 3.9 ˜100% acetylationPEG-C3-CD 18.07 4.8 None PEG-C3-AcCD 17.86 4.8  ˜80% acetylationPEG-L8-CD (A) 18.12 4.6 None PEG-L8-AcCD (A) 17.98 4.6  ˜95% acetylationPEG-L8-CD (B) 18.43 5.9 None PEG-L8-AcCD (B) 18.08 5.9  ˜84% acetylationPEG-L8-CD (C) 18.71 5.4 None PEG-L8-AcCD (C) 18.53 5.4 ˜100% acetylationPEG-GFLG-DECD 2.5  ˜67% ethylation PEG-L8-DECD 18.0  3.9  ˜67%ethylation PEG-C3-BnCD 4.5  ˜80% Butyrylation

EXAMPLE 7 Preparation of Paclitaxel Complexes with CD Polymers or CDMonomers

[0097] (A) Co-Dissolving Method: This Method is Suitable for allComplexes with Water Soluble Polymers

[0098] The aqueous solution of polymer (or monomer controls) (usuallyabout 100 mg/ml) was mixed with equal volume (usually 40 to 2000 ul) ofthe paclitaxel solution (C_(paclitael)=0.1 to 8.0 mg/ml in methanol).The mixture was incubated at room temperature for about half an hour.Then the solvents were removed in a centrifuge concentrator at roomtemperature. The concentrated syrup or wax solid was reconstituted byadding water or PBS buffer to the original volume. The mixture wasusually a clear or slightly cloudy solution after 30 minutes ofreconstitution. The un-dissolved paclitaxel particles were removedeither by ultra-filtration (0.2 um filter) or by centrifugation (20minutes at 20,800 g and room temperature). The paclitaxel concentrationin the clear supernatant was quantified by UV absorbance at 290 nm byusing the corresponding cyclodextrin polymer solution as the backgroundcalibration.

[0099] (B) Dialysis Method: This Method is Suitable for the Preparationof all Paclitaxel/Polymer Complex Solutions:

[0100] The methanol solution of the polymer (usually 1100 mg/ml) wasmixed with equal volume (1100 ul) of paclitaxel solution (1 to 3 mg/mlin methanol). The clear mixture was incubated at room temperature forabout half an hour at room temperature, followed by dialysis(MWCO=12,000) overnight against 2 L water. The dialysis solution wasusually a clear solution. Trace amounts of paclitaxel particles wereremoved either by ultra-filtration (0.2 um filter) or by centrifugation(20 minutes at 20,800 g and room temperature). The clear solution wasstored at 4° C. or below.

EXAMPLE 8

[0101] Preparation of Antisense Oligonucleotide/CD-Polymer Complexes

[0102] Cyclodextrin PEG polymers (50 mg/ml) were mixed with a certainamount of a 21-mer-fluorescent labeled oligonucleotide in 20 mM Tris-HClbuffer (pH=7.4). The solutions were dried in a Speed-Vac, followed byreconstitution using the same amount of water. The DNA/polymer complexesin the solution were analyzed using 1% agarose gel in pH=7.4 TAE buffer.TABLE 3 Comparison of Paclitaxel or Oligonucleotide Loading by some ofthe co-polymers as compared with other available CD derivatives CDmoiety/ Paclitaxel loading Oligonucleotide loading (mg/50 mg (mg/50 mgPolymer Polymer polymer*) polymer*) PEG-ss-CD 3.9 <0.05   ND**PEG-ss-AcCD 3.9 0.8 ND PEG-C3-CD 4.8 <0.05 ND PEG-C3-AcCD 4.8 2.0 NDPEG-L8-CD (A) 4.6 <0.05 ND PEG-L8-AcCD (A) 4.6 2.6 ND PEG-L8-CD (B) 5.9<0.05 ND PEG-L8-AcCD (B) 5.9 2.9 ND PEG-ss-DECD 1.5 0.4 0.06PEG-GFLG-DECD 2.5 3.0 0.2 PEG-C3-DECD 2.6 <1.0 0.15 PEG-L8-DECD 3.9 <1.00.2 Controls HP-CD (from Sigma) <0.05 (SBE)₇-CD (from Cydex) <0.05 DM-CD(from Sigma) ˜0.2 (at day 1) EP-CD (from Sigma) <0.05

EXAMPLE 9

[0103] Stability of Taxol/CD Complexes in 50% Serum or After 10 FoldDilution in PBS:

[0104] (A) Stability in 50% fetal bovine serum: Taxol/PEG-L8-AcCD (2.0mg/50 mg in 1.0 ml PBS buffer) or Taxol/DMCD (0.5 mg/50 mg in PBSbuffer) complex solutions were prepared as describe in method A ofexample 7. Fifty micro liters of the complex solutions were diluted withequal volume of fetal bovine serum respectively. Both mixtures werecentrifuged at 20,8000 g at room temperature after incubation at roomtemperature for 2 hours, 21 hours, 49 hours and 144 hours, respectively.The Taxol concentration in each supernatant was quantified by measuringthe UV absorption at 230 nm.

[0105] (B) Stability after 10 fold dilution with PBS: Taxol/PEG-L8-AcCD(2.0 mg/50 mg in 1.0 ml PBS buffer) or Taxol/DMCD (0.5 mg/50 mg in PBSbuffer) complex solutions were prepared as describe in example 10. Fiftymicro liters of the complex solutions were diluted with 450 micro literof PBS buffer, respectively. Both mixtures were centrifuged at 20,8000 gat room temperature after incubated room temperature for 2 hours, 21hours, 49 hours and 144 hours. The Taxol concentration in eachsupernatant was quantified by measuring the UV absorption at 230 nm.TABLE 4 Summary of the Stability Test of Pacitaxel/PEG-L8-AcCD andPacitaxel/DMCD complexes in 50% Serum or after dilution with PBSRemaining paclitaxel % in diluted solution Time PEG-L8-AcCD PEG-L8-AcCDDMCD 50% DMCD 10X (hour) 50% Serum 10X PBS Serum PBS  0 100  100 100100   2 100  100 100 86 21 97 100 100 13 49 92  99 100 144  84  79 100

EXAMPLE 10

[0106] Release of Paclitaxel from the Paclitaxel/PEG-L8-AcCD Complexesand the Cytotoxicity of the Free Co-Polymers

[0107] The efficient release of the free paclitaxel from its PEG-L8-AcCDcomplex was confirmed by the cytotoxicity of the complexes. Similar IC₅₀values were obtained for both paclitaxel/PEG-L8-AcCD complex formulation(in this invention) and current commercial Paclitaxel/Cremophorformulation (Taxol, Bristol-Myers Squibb) in all three tested cell linesas determined by modified MTT assay as described below. But PEG-L8-AcCDalone showed no detectable cytotoxicity at the highest testingconcentration while cremophor killed half of the cells at aconcentration of about 0.5 mg/ml (Table 5):

[0108] 1. Cells were plated at about 5,000 cells/well in 96-well platesin 0.1 ml medium and incubated at 37° C. for 24 hours;

[0109] 2. Remove the old medium, add 80 ul of fresh media to each well;

[0110] 3. Add 20 ul of sample solutions to each well (5×seriallydiluted, at least 8 concentrations for each sample)

[0111] 4. The cells were incubated 3 or 4 days;

[0112] 5. The media was removed. Added 80 ul of fresh media with 20 ulof MTS solution (Promega CellTiter 96 Aqueous One Solution Reagent#G358A). Incubate 37° C. for 2 to 4 hours;

[0113] 6. Read absorbance at 490 nm on plate reader

[0114] 7. Calculate the IC₅₀ using cell free well as blank control anddrug free well as 100% viability control. TABLE 5 Comparison of theIC₅₀* of different Taxol formulations and carrier controls in threedifferent cell lines IC₅₀ (ng/ml) Formulations Hela HT1080 MCF7Paclitaxel/ 3.0 2.0 2.0 PEG-L8-AcCD Paclitaxel/ 3.0 4.0 2.0 CremophorCremophor 500,000 500,000 500,000 PEG-L8-AcCD >10,000,000 >10,000,000>10,000,000

EXAMPLE 11

[0115] Hemolysis Activity of the Co-Polymers and Their PossibleBiodegradation Products:

[0116] To further investigate the cytotoxicity of our polymers and theirpossible biodegradation products, their hemolysis effects were tested onfresh human blood cells in comparison with commercial CD monomers asdescribe below. The degree of hemolysis was reported as a percentage ofthe total efflux of hemoglobin in distilled water (Table 6)

[0117] 1. Red blood cells were isolated from whole human blood bycentrifugation at 1000 g for 10 minutes.

[0118] 2. The plasma was removed and the red blood cells re-suspended innormal buffed saline (PBS, 0.154 M sodium chloride and 0.01 M phosphate,pH=7.4). The red blood cells were pelleted by centrifugation (1000 g for10 minutes).

[0119] 3. Step 2 was repeated twice to remove the heme released fromdamaged cells.

[0120] 4. The final pellet was diluted with PBS to give a hematocrit ofapproximately 12 (or 5%) as determined by centrifugal sedimentation.

[0121] 5.2 ml of polymer or cyclodextrin solutions of a series ofconcentrations from 0 to 50 mg/ml in PBS buffer) equilibrated at 37° C.in PBS buffer were equilibrated at 37° C. To this was added 100 ul of ared blood cell suspension followed by mixing of the sample with gentleinversion. The samples were incubated for 30 minutes at 37° C.

[0122] 6. The intact cells and cellular debris were pelleted bycentrifugation at 1000 g for 5 min. The supernatant was analyzedspectrophotometrically at 543 nm for released heme. TABLE 6 Comparisonof Hemolysis activities of different PEG-CD polymers and their precursormonomers with commercial CD derivatives. Comopmers or Hemolysis CDmonomers (HC₅₀, mM) PEG-L8-AcCD ND PEG-L8-DECD ND PEG-L8-CD ND CD-L8-NH225 (SBE)₇-CD ND DM-CD 1.0 βCD 4.0 HPβCD 35

[0123] The above data show that the novel PEG-CD polymers of the presentinvention have great potential to be used as safe drug carriers forpaclitaxel (Table 3, Table 4, Table 5 and Table 6). In the presence of50 mg/ml of the polymers, the paclitaxel can be dissolved in water at aconcentration of at least 2.2 mg/ml, which is more than a 10,000 foldincrease in free paclitaxel water solubility, and at least 1,000 and 20times better than that of hydroxylpropyl-β-cyclodextrin (HPCD) andmethyl-β-cyclodextrin (DMCD), respectively, under similar conditions[Sharma et al. J Pharm Sci, 84 (10), 1223-30 (1995)]. This dramaticsolubility increase may due to a combination of at least the followingthree factors: 1) increased local concentration of CD moieties; 2)increased binding constant by cooperation the structure of paclitaxelhas three phenyl groups around a large, fused taxane ring system); and3) extra hydrophobic interactions outside the CD cavities.

[0124] As expected, after being conjugated to PEG polymer, the toxicityof β-cyclodextrin was significantly reduced. No cytotoxicity wasdetected on all the cyclodextrin pendent PEG polymers as identified onMTT and hemolysis assays (Table 5 and Table 6). Even the monomer(building block) was much less toxic than natural β-cyclodextrin. Onanother hand, because the weight ratios of CD moieties in our currentco-polymer were only less than 25% as determined by ¹H-NMR, the actualCD concentration in our experimental concentration (50 mg co-polymer/mlwater) was less than 12.5 mg/ml. In another words, the weight ratio ofcyclodextrin: Paclitaxel moiety was less than 6:1 in the current polymercomplexes. Therefore, the co-polymers with non-biodegradable linkers arevery safe drug carriers with very efficient drug release characteristics(Table 5). Additionally, the biodegradable linkage may also beacceptable as necessary to accelerate drug release.

[0125] The above Examples are presented for illustrative purposes onlyand are not intended, and should not be constructed to limit theinvention in any manner. Various modifications of the compounds andmethods of the invention may be made without departing from the spiritor scope thereof and it is to be understood that the invention isintended to be limited only as defined in the appended claims.

We claim:
 1. A cyclodextrin grafted biocompatible polymer having theformula 1:

wherein P is a biocompatible hydrophilic polymer backbone having amolecular weight range from 2,000 to 1,000,000 Daltons; R′ is H or atargeting moiety; X is a linker having the formula -Q-Z-Q′- wherein Q iscovalently bonded to the hydrophilic polymer chain either directly or bymeans of a pendant alkyl or other functional group and Q′ is covalentlybonded to the cylodextrin at the 2, 3 or 6 position thereby replacingeither OR₁, OR₂ or OR₃ group respectively; Q and Q′ are independentlymembers selected from the group consisting of NR₄, S, O, CO, CONH, andCOO; Z is a member selected from the group consisting of an alkylenedisulfide, [—(CH₂)_(a)S—S(CH₂)_(a)—], alkylene [—(CH₂)_(a)—], alkyleneoxide (—[(CH₂)_(a)O]_(b)(CH₂)_(a)—), or a short chained peptide where ais an integer of 1 to 10 and b is an integer of 1 to 20; R₁, R₂, R₃ andR₄ are independently members selected from the group consisting of H,alkyl (C_(n′)H_(2n′+1)), alkenyl (C_(n′+1)H_(2(n′+1)−1)) or acyl(C_(n′)H_(2n′+1)CO) where n′ is an integer of 1 to 16; q is an integerof 5, 6 or 7; and w is an integer such that each polymer backbonecontains between 1.5 and 30 cyclodextrin moieties per 20 KD of polymerbackbone.
 2. The cyclodextrin grafted biocompatible polymer of claim 1wherein the biocompatible polymer is a member selected from the groupconsisting of polyethylene glycol (PEG),N-(2-hydroxypropyl)methacrylamide polymer (HPMA), polyethylenimine(PEI), polylysine, derivatives thereof and polymers thereof.
 3. Thecyclodextrin grafted biocompatible polymer of claim 2 wherein eachpolymer backbone contains between 2 and 15 cyclodextrin moieties per 20KD of polymer backbone.
 4. The cyclodextrin grafted biocompatiblepolymer of claim 3 wherein the biocompatible polymer has a molecularweight of between about 5,000 and 70,000.
 5. The cyclodextrin graftedbiocompatible polymer of claim 4 where Q is C(O)NH, Q′ is NR₄ and a is2.
 6. The cyclodextrin grafted biocompatible polymer of claim 4 where Zis —(CH₂)₂S—S(CH₂)₂—; R₄ is C₂H₅, R₁ is C₂H₅, R₂ is H and R₃ is C₂H₅. 7.A cyclodextrin grafted biocompatible polymer having the formula 2

wherein R′ is H or a targeting moiety; X is a linker having the formula-Q-Z-Q′- wherein Q is covalently bonded to the hydrophilic polymer chaineither directly or by means of a pendant alkyl or other functional groupand Q′ is covalently bonded to the cylodextrin at the 2, 3 or 6 positionthereby replacing either OR₁, OR₂ or OR₃ group respectively; Q and Q′are independently members selected from the group consisting of NR₄, S,O, CO, CONH, and COO; Z is a member selected from the group consistingof an alkylene disulfide, [—(CH₂)_(a)S—S(CH₂)_(a)—], alkylene[—(CH₂)_(a)—], alkylene oxide (—[(CH₂)_(a)O]_(b)(CH₂)_(a)—), or a shortchained peptide where a is an integer of 1 to 10 and b is an integer of1 to 20; R₁, R₂, R₃ and R₄ are independently members selected from thegroup consisting of H, alkyl (C_(n′)H_(2n′+1)), alkenyl(C_(n′+1)H_(2(n′+1)−1)) or acyl (C_(n′)H_(2n′+1)CO) where n′ is aninteger of 1 to 16; q is an integer of 5, 6 or 7; w is an integer suchas to provide between 2 and 15 cyclodextrin units per 20 KD PEG backbonechain, and m and n are integers sufficient that when combined with wthey represent a polyethylene oxide polymeric chain having the molecularweight of 5,000 to 70,000 with the proviso that monomeric units on thebiocompatible polymer backbone containing the grafted cyclodextrin unitsrepresented by w do not have to be consecutively joined but may berandomly or uniformly distributed along polymer backbone.
 8. Thecyclodextrin grafted biocompatible polymer of claim 7 where Q is C(O)NH,Q′ is NR₄ and a is
 2. 9. The cyclodextrin grafted biocompatible polymerof claim 7 where Z is —(CH₂)₂S—S(CH₂)₂; R₄ is C₂H₅, R₁ is C₂H₅, R₂ is Hand R₃ is C₂H₅.
 10. A cyclodextrin grafted biocompatible polymer havingthe formula 3

wherein R′ is H or a targeting moiety; Q is covalently bonded to thehydrophilic polymer chain either directly or by means of a pendant alkylor other functional group and Q′ is covalently bonded to the cylodextrinat the 2, 3 or 6 position thereby replacing either OR₁, OR₂ or OR₃ grouprespectively; Q and Q′ are independently members selected from the groupconsisting of NR₄, S, O, CO, CONH, and COO; Z is a member selected fromthe group consisting of an alkylene disulfide,[—(CH₂)_(a)S—S(CH₂)_(a)—], alkylene [—(CH₂)_(a)—], alkylene oxide(—[(CH₂)_(a)O]_(b)(CH₂)_(a)—), or a short chained peptide where a is aninteger of 1 to 10 and b is an integer of 1 to 20; R₁, R₂, R₃ and R₄ areindependently members selected from the group consisting of H, alkyl(C_(n′)H_(2n′+1)), alkenyl (C_(n′+1)H_(2(n′+1)−1)) or acyl(C_(n′)H_(2n′+1)CO) where n′ is an integer of 1 to 16; q is an integerof 5, 6 or 7; w is an integer such as to provide between 2 and 15cyclodextrin units per 20 KD PEG backbone chain, and m and n areintegers sufficient that when combined with w they represent apolyethylene oxide polymeric chain having the molecular weight of 5,000to 70,000 with the proviso that monomeric units on the hydrophilicpolymer chain containing the cyclodextrin units represented by w do nothave to be consecutively joined but may be randomly or uniformlydistributed along polymer chain.
 11. The cyclodextrin graftedbiocompatible polymer of claim 10 where Q is C(O)NH, Q′ is NR₄ and a is2.
 12. The cyclodextrin grafted biocompatible polymer of claim 10 whereZ is —(CH₂)₂S—S(CH₂)₂—; R₄ is C₂H₅, R₁ is C₂H₅, R₂ is H and R₃ is C₂H₅.13. A composition comprising a cyclodextrin grafted biocompatiblepolymer of claim 1 and an active agent.
 14. The composition of claim 13wherein the active agent is a hydrophobic drug, a protein or peptidedrug, a nucleic acid or an oligo nucleotide
 15. The composition of claim13 wherein the active agent is paclitaxel.
 16. A composition comprisinga cyclodextrin grafted biocompatible polymer of claim 7 and an activeagent.
 17. The composition of claim 16 wherein the active agent is ahydrophobic drug, a protein or peptide drug, a nucleic acid or an oligonucleotide.
 18. The composition of claim 16 wherein the active agent ispaclitaxel.
 19. A composition comprising a cyclodextrin graftedbiocompatible polymer of claim 10 and an active agent.
 20. Thecomposition of claim 19 wherein the active agent is a hydrophobic drug,a protein or peptide drug, a nucleic acid or an oligo nucleotide
 21. Thecomposition of claim 19 wherein the active agent is paclitaxel.
 22. Amethod for effectively delivery of an active agent to a warm bloodedanimal comprising administering an effective amount of a composition ofclaim 13 to said warm blooded animal.
 23. The method of claim 22 whereinthe active agent is a hydrophobic drug, a protein or peptide drug, anucleic acid or an oligo nucleotide
 24. The method of claim 22 whereinthe active agent is paclitaxel.
 25. A method for effectively delivery ofan active agent to a warm blooded animal comprising administering aneffective amount of a composition of claim 16 to said warm bloodedanimal.
 26. The method of claim 25 wherein the active agent is ahydrophobic drug, a protein or peptide drug, a nucleic acid or an oligonucleotide
 27. The method of claim 25 wherein the active agent ispaclitaxel.
 28. A method for effectively delivery of an active agent toa warm blooded animal comprising administering an effective amount of acomposition of claim 19 to said warm blooded animal.
 29. The method ofclaim 28 wherein the active agent is a hydrophobic drug, a protein orpeptide drug, a nucleic acid or an oligo nucleotide
 30. The method ofclaim 28 wherein the active agent is paclitaxel.